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Journal of Bacteriology, May 1999, p. 3018-3024, Vol. 181, No. 10
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Alterations in Protein Expression Caused by the
hha Mutation in Escherichia coli: Influence of
Growth Medium Osmolarity
Carlos
Balsalobre,1,
Jörgen
Johansson,2
Bernt
Eric
Uhlin,2
Antonio
Juárez,1 and
Francisco J.
Muñoa1,*
Departamento de Microbiología,
Universidad de Barcelona, Barcelona 08028, Spain,1 and Department of Microbiology,
Umeå University, S-90187 Umeå, Sweden2
Received 30 November 1998/Accepted 1 March 1999
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ABSTRACT |
The Hha protein belongs to a new family of regulators involved in
the environmental regulation of virulence factors. The aim of this work
was to study the effect of the hha mutation on the overall
protein pattern of Escherichia coli cells by
two-dimensional polyacrylamide gel electrophoresis. The growth medium
osmolarity clearly influenced the effect of the hha
mutation. The number of proteins whose expression was altered in
hha cells, compared with wild-type cells, was three times
larger at a high osmolarity than at a low osmolarity. Among the
proteins whose expression was modified by the hha allele,
both OmpA and protein IIAGlc of the phosphotransferase
system could be identified. As this latter enzyme participates in the
regulation of the synthesis of cyclic AMP and hence influences the
catabolite repression system, we tested whether the expression of the
lacZ gene was also modified in hha mutants.
This was the case, suggesting that at least some of the pleiotropic
effects of the hha mutation could be caused by its effect
on the catabolite repression system.
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INTRODUCTION |
The chromosomal hha gene
of Escherichia coli was identified in a search for mutants
of E. coli 5K that overproduced the toxin alpha-hemolysin
from a plasmid-encoded hemolytic operon (11). The product of
the hha gene is the 8.6-kDa Hha protein (25), which is highly homologous (82%) to the Yersinia
enterocolitica YmoA protein (10). The YmoA protein is a
temperature-dependent modulator of the expression of some virulence
factors in this species (8). Hha participates in the
osmolarity-dependent expression of hemolysin (7, 22). Both
the ymoA mutation (8) and the hha
mutation (11, 22) are pleiotropic. Effects on
environmentally dependent modulation of gene expression and pleiotropy
are properties of a well-characterized class of mutants, the
hns mutants (4, 12, 14, 17; see reference
1 for a review). Because the product of the
hns gene is the nucleoid-associated protein H-NS, it was
suggested that Hha and YmoA are also nucleoid-associated proteins. With
respect to Hha, several lines of experimental evidence support this
hypothesis: hha mutants show alterations in the topology of
a reporter plasmid (7), overexpression of Hha increases the
frequency of transposition of insertion elements in the E. coli chromosome (2, 19), and Hha is a DNA-binding
protein (17a).
The aim of this work was to better characterize the pleiotropic effect
of the hha mutation with respect to its
osmolarity-modulating role. Two-dimensional (2-D) polyacrylamide gel
electrophoresis (PAGE) analysis of total cellular extracts obtained
from wild-type E. coli 5K and from its hha
derivative, strain Hha-3, after growth in media of low or high
osmolarity was done. 2-D PAGE protein maps of E. coli K-12
(34) were used to identify some of the proteins whose
expression was found to be altered by the mutation.
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MATERIALS AND METHODS |
Bacterial strains, plasmids, and media.
E. coli 5K,
Hha-3 (hha::Tn5) (11),
CCB21, and CCB21H (hha::Tn5)
(22) have been previously described. Plasmid pCB26R contains a translational hlyC::lacZ fusion that
generates an in-frame gene fusion that is transcribed under the control
of the hly promoter and gives rise to a hybrid HlyC-LacZ
protein. Its construction has already been described (21).
Synthetic medium Rich-MOPS (24) was used for the growth of
strains. Glucose (0.4%) was included as a carbon source in all the
experiments in which Rich-MOPS was used. NaCl was added to the medium
for high-osmolarity experiments. Cultures were incubated at 37°C.
Protein labelling.
When the optical density at 600 nm
(OD600) of the cultures reached 0.5, a 1-ml sample was
removed and pulse-chased for 15 min with [35S]methionine.
The procedure was used for both one-dimensional sodium dodecyl sulfate
(SDS)-polyacrylamide gel electrophoresis (PAGE) and two-dimensional
(2-D) PAGE analyses of whole-cell extracts.
Gel electrophoresis and Western blotting.
SDS-PAGE (10%
polyacrylamide) was done as described by Laemmli (16). For
Western blot analysis, polyclonal rabbit serum raised against OmpA
(13) was used at a dilution of 1:5,000 from a stock
containing 75 mg ml
1.
2-D SDS-PAGE.
Preparation of cell extracts for 2-D PAGE and
the 2-D PAGE itself were performed by the method of O'Farrell
(26) with modifications (34). We used an
Investigator 2-D Electrophoresis System (Millipore Corporation); all
reagents were used as described in the product manual. Carrier
ampholytes for the first dimension were in the range of pH 3 to 10. An
SDS-12.5% polyacrylamide gel was used for the second dimension. The
amounts of extracts loaded into the gels were adjusted after
measurement of the radioactivity incorporated by each sample.
Comparative studies of protein spot intensities were done by analysis
of the gels with a Milligen Bioimage 2-D analysis package.
Measurement of 
-galactosidase.
Strain 5K containing
plasmid pCB26R was cultured in 0.4 M NaCl-Rich-MOPS and in 0 M
NaCl-Rich-MOPS. Strains CCB21 and CCB21H were grown in
low-glucose-containing Luria broth (LB) (tryptone yeast broth; Svenska
LabFab) either without NaCl or with 0.5 M NaCl and supplemented with
0.2% lactose. When cultures reached an OD600 of 0.5, samples were taken and -galactosidase activities in permeabilized
cells were assayed as described by Miller (20). When needed,
cyclic AMP (cAMP) was added at a final concentration of 3 mM.
SDS-PAGE analysis of whole membrane proteins.
Purification
of the whole membrane fraction was done as follows. Cultures (400 ml)
of strains 5K and Hha-3 grown in LB either without NaCl or with 0.5 M
NaCl were harvested at an OD600 of 0.5. Cells were pelleted
and resuspended in 10 ml of buffer A (10 mM Tris-HCl, 5 mM
MgCl2 [pH 7.6]). This step was done twice. Cells were
disrupted by use of a French press (SLM Instruments, Inc.) at 900 lb/in2. This step was also done twice. Cell extracts were
then centrifuged at 16,000 rpm for 2 min to eliminate unbroken cells,
and the supernatant was centrifuged at 22,300 rpm for 2 h (Beckman
L8-M ultracentrifuge, rotor type 70.1 Ti). The pellet was finally
resuspended in 2 ml of buffer A. The protein concentration was
measured, 5 µg of each sample was mixed with standard protein sample
buffer (containing SDS and -mercaptoethanol), and the mixture was
heated at 100°C for 5 min and loaded onto an SDS-10% polyacrylamide
gel. This procedure allows the isolation of proteins located in both membranes.
Transport assay.
The transport assay was basically carried
out as described by Buhr et al. (6), with slight
modifications. Cells were grown by diluting an overnight culture
200-fold in 100 ml of Rich-MOPS. When the culture had reached an
OD600 of 0.5, cells from 20 ml of culture were washed in
ice-cold Rich-MOPS without glucose and resuspended in 1 ml of the same
medium. The cell suspension was preincubated for 10 min at room
temperature with aeration. [U-14C]glucose was then added
to a final concentration of 400 µM (diluted to 1,000 dpm/nmol;
Amersham). Aliquots (100 µl) were removed after 10, 20, 30, 50, 100, and 300 s, quenched by dilution in 8 ml of ice-cold Rich-MOPS
complemented with 400 µM unlabelled glucose, and immediately applied
to glass fiber filters (GF/F; Whatman) under suction. The filters were
washed with 20 ml of ice-cold 0.9% NaCl, and counts were determined
with 5 ml of Optiphase (Wallac). The glucose uptake rate was calculated
as nanomoles minute1 milligram1 (dry
weight) from the linear part of the uptake curve.
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RESULTS AND DISCUSSION |
Growth kinetics analysis.
Our previous work on the influence
of growth medium osmolarity on the expression of alpha-hemolysin in
strains 5K and Hha-3 was carried out by growing cells in LB without
NaCl (low-osmolarity medium) and in LB containing 0.5 M NaCl
(high-osmolarity medium) (7, 22). No differences in growth
rates were detected between these two strains for a given medium
(unpublished data). Pulse-labelling experiments required a defined
synthetic medium (Rich-MOPS); therefore, additional growth kinetics
comparisons of the two strains were necessary. Standard Rich-MOPS
either without NaCl or with 0.1 to 0.5 M NaCl was used to study the
growth kinetics. Differences in the growth rates of the two strains
were detected only when the cells were cultured at 0.5 M NaCl (data not
shown). In accordance with these results, Rich-MOPS with 0.4 M NaCl was
used in experiments requiring high-osmolarity conditions, while
Rich-MOPS without NaCl was used in experiments requiring low-osmolarity conditions.
Previous work with strain 5K cultured in LB without NaCl and in LB
containing 0.5 M NaCl was carried out by us to analyze the influence of
the osmolarity of the medium on hemolysin expression. Low osmolarity
increased hemolysin expression (21). In that study,
transcription of the hemolysin operon was monitored by measuring
-galactosidase activity in cells harboring plasmid pCB26R, which
contains an in-frame gene fusion between the hlyC gene of
the hemolysin operon and the lacZ gene. In order to confirm that growth in Rich-MOPS was equivalent to growth in LB with respect to
hemolysin expression, we measured the -galactosidase activities of
strain 5K carrying plasmid pCB26R in 0.4 M NaCl-Rich-MOPS and in
Rich-MOPS without added NaCl (0 M NaCl). The activity at low osmolarity
was about twice the activity at high osmolarity (1,592 versus 799 Miller units). These results agreed with those obtained when LB was
used (21) and indicated that Rich-MOPS could be used to
monitor the effect of different concentrations of NaCl on the pattern
of proteins expressed by the strains that we studied.
Electrophoretic analysis.
As hha mutants exhibit a
pleiotropic phenotype, we tried to observe changes in the overall
pattern of protein synthesis due to hha mutations. An
initial comparison between the parental and mutant strains was carried
out with cultures grown at different osmolarities. Figure
1 shows the autoradiogram obtained from
an SDS-PAGE analysis of total cellular proteins after radioactive labelling. Only minor differences between the parental strain 5K and
the hha mutant were observed when the cells were grown at
low osmolarity. In contrast, several distinct differences were observed
when the cells were cultured at high osmolarity.
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FIG. 1.
Autoradiogram from SDS-10% PAGE analysis. Total
cellular protein extracts from strains 5K (lanes 1 and 3) and Hha-3
(lanes 2 and 4) after growth at low (lanes 1 and 2) and high (lanes 3 and 4) osmolarities were analyzed (see text for details). Arrowheads
indicate major differences.
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To better characterize such differences in the protein synthesis
pattern, we analyzed total cellular extracts by 2-D PAGE
and searched
for proteins whose expression increased or decreased
in strain Hha-3
with respect to the parental strain. Figure
2 shows the results obtained for both
strains cultured either at
low or at high osmolarity. Although the
amount of protein loaded
onto the gels was adjusted by the
radioactivity incorporated into
each sample, the autoradiograms in Fig.
2 are not completely equivalent,
and slight background differences are
apparent. For this reason
and for a given pair of gels, we decided to
highlight on Fig.
2 only those spots that were clearly different in
both strains.
As shown in Fig.
2A and B (low-osmolarity medium), three
proteins
were present in the wild-type strain, four were present in the
mutant, and two were present in both but showed higher expression
in
the mutant strain. In Fig.
2C and D (high-osmolarity medium),
4 proteins were present in the wild-type strain, 5 were present
in the
mutant, and 17 were present in both, 11 with higher expression
in the
wild-type strain and 6 with higher expression in the mutant
strain. The
lack of the Hha protein not only increased the expression
of some
genes, as it does with the alpha-hemolysin determinant
(
11),
but also decreased the expression of others.
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FIG. 2.
Autoradiograms from 2-D PAGE separation of proteins in
total cellular extracts from E. coli 5K and Hha-3 after
growth in media of different osmolarities. In each panel, the gel is
oriented with the more acidic proteins on the left. (A) Wild-type
strain 5K at 0 M NaCl. (B) Mutant strain Hha-3 at 0 M NaCl. (C) Strain
5K at 0.4 M NaCl. (D) Strain Hha-3 at 0.4 M NaCl. For a given
osmolarity, proteins in circles are those detected in strain 5K but not
in strain Hha-3, while proteins in boxes are those detected in strain
Hha-3 but not in strain 5K. Arrows indicate proteins present in both
strains but at different intensities. Proteins in triangles are two
osmoregulated porins (see text for details).
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As the absence of Hha clearly seemed more significant when the cells
were cultured at high osmolarity, we further analyzed
some of the
differences observed in the high-osmolarity gels.
Table
1 shows the densitometric values
calculated for 11 of the
highlighted spots in Fig.
2C and D. By using
the gene-protein
database for
E. coli (
34), we
were able to locate the alpha-numeric
coordinates of five of the
affected proteins and to identify the
genes for two of them,
ompA and
crr, which encode proteins OmpA
and
enzyme IIA
Glc of the phosphotransferase system,
respectively. A third gene
was identified from an Internet database
(
23a) as
ahpC, which
encodes the alkyl
hydroperoxide reductase that is a member of
the superoxide stress
regulon.
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TABLE 1.
Spot intensity comparison between the protein patterns of
strains 5K (wild type) and Hha-3 (hha mutant) from Fig.
2C and D
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Comparison of the spots detected by 2-D PAGE analysis with the
reference map from Van Bogelen et al. (
34) gives only an
indication of what certain proteins are; thus, they have to be
identified by other means. For that reason and given the relevant
role
of proteins OmpA and enzyme IIA
Glc, we further investigated
if the expression of both proteins is
indeed affected by
hha mutations.
Effect of an hha mutation on the expression of
OmpA.
OmpA is one of the most abundant proteins of the E. coli envelope. It maintains the integrity of the E. coli outer membrane (33), it functions as a mediator in
F-dependent conjugation (32), it stimulates a strong
antibody response (29), and it may play an important role in
virulence
an OmpA-deficient mutant of E. coli K-1 showed
reduced virulence in a rat model of bacteremia (18). One of
the four OmpA isoforms that can be detected by 2-D analysis
(34) corresponds to one of the proteins identified as having
altered expression in strain Hha-3 grown at high osmolarity. It
corresponds to the alpha-numeric coordinates F028.0. Quantification of
its expression showed that it decreased by 59% in strain Hha-3 in
comparison with parental strain 5K. In order to confirm that the spot
was indeed OmpA, we used antibodies against OmpA. Again, 2-D PAGE
analysis of the extracts was carried out, proteins being transferred to
membranes and immunoblotted. As shown in Fig.
3, the antibodies recognized three of the
four OmpA isoforms and did not recognize other proteins in the 2-D
gels. The fact that one of the three spots recognized corresponds to a
spot highlighted in Fig. 2 confirms it as being OmpA. It is important
to note that, when one is looking at the immunodetected spots,
differences in intensity seem to be smaller than those detected by
autoradiograms, but one must consider that, with the conditions used,
immunodetection is qualitative rather than quantitative.
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FIG. 3.
Immunodetection of OmpA among the spots obtained by 2-D
PAGE. (A and C) Immunodetection of the OmpA isoforms (designated I, II,
and III) in cell extracts obtained, respectively, from wild-type and
hha strains grown at high osmolarity. (B and D)
Magnification of the area from the original 2-D gels used to detect
OmpA.
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OmpA is an exported protein, like other proteins whose final
destination is located outside the cytoplasm. Most of the exported
E. coli proteins share a general secretory machinery for
leaving
the cytoplasm and, for that reason, the effect of the
hha mutation
on OmpA expression could be the consequence of
a more generalized
influence on the mechanisms of protein export to the
cell envelope.
This seemed not to be the case, because two
osmoregulated porins,
OmpC and OmpF (
9), that were
identified in Fig.
2 were not
affected by the
hha mutation.
In order to confirm that the effect of the
hha mutation was
specific for OmpA, envelope proteins from both wild-type and
hha mutant strains were analyzed by SDS-PAGE.
Immunodetection of OmpA
was also carried out to allow for OmpA
identification. As shown
in Fig.
4, only
OmpA was clearly affected by the mutation. At
high osmolarity, slight
intensity differences were also apparent
for a band that was located
just above OmpA and that was also
recognized by the anti-OmpA
antibodies (Fig.
4). As the antibodies
previously had been proven to be
very specific for detecting OmpA
in 2-D PAGE (Fig.
3) and because of
the sizes of the immunodetected
bands (35.2 and 37.2 kDa), we reasoned
that they could be the
mature and the precursor forms of OmpA.
According to these results,
it seems that the effect of the
hha allele is specific for OmpA
expression and is not a more
general effect on a wider range of
envelope proteins, at least not
those detected by SDS-PAGE. Whether
or not this effect on OmpA
expression is important for the structural
and/or physiological
characteristics of the
hha mutants is beyond
the scope of
this work.
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FIG. 4.
Analysis of cell envelope proteins in wild-type and
hha mutant E. coli strains grown at low and high
osmolarities. (A) Western blot analysis with OmpA-specific antibodies
of the envelope proteins of strain 5K cultured in standard LB. (B)
Coomassie blue-stained gel from SDS-PAGE analysis of envelope proteins
of strains 5K (lanes 1 and 3) and Hha-3 (lanes 2 and 4) grown in LB
with 0.5 M NaCl (lanes 1 and 2) and in LB without NaCl (lanes 3 and 4).
P and M, precursor and mature forms, respectively, of OmpA.
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Physiological effects of enzyme IIAGlc levels altered
by an hha mutation.
The second identified protein
corresponds to the alpha-numeric coordinates B018.7. This protein is
enzyme IIAGlc of the phosphoenolpyruvate:glucose
phosphotransferase system (PTS) and is encoded by the crr
gene. Its expression was decreased by 81% in strain Hha-3 in
comparison with the parental strain. This enzyme plays a crucial role
in the E. coli carbohydrate phosphotransferase system. It is
one of the enzymes involved in the pathway for the uptake of glucose,
it regulates the activity of the adenylate cyclase, and it is also
responsible for the inducer exclusion of non-PTS carbohydrates
(28). It is well known that the phosphorylation state of
enzyme IIAGlc regulates the cAMP biosynthetic enzyme,
adenylate cyclase (31). cAMP can bind to the cAMP receptor
protein (CRP) to form the cAMP-CRP complex, which is a regulatory
factor for the transcription of several hundred genes (5,
15). Taking into consideration the important role of the enzyme
IIAGlc in E. coli metabolism, we decided to use
a physiological approach to confirm that the identified protein was
enzyme IIAGlc. We studied the effect of the hha
mutation on the expression of a gene that is catabolite repression
sensitive, the lacZ gene. We also examined functional
implications with respect to effects on inducer exclusion and on
glucose uptake.
(i) Effect on cAMP-dependent gene expression.
Variations in
the expression of crr influence the activity of adenylate
cyclase and therefore modify the level of cAMP (27). This
effect could be monitored by measuring the expression of genes
regulated by cAMP-CRP. With this aim, we decided to analyze if the
expression of the lac operon was affected by the
hha mutation. In this experiment, we used the isogenic
E. coli Lac+ strains CCB21 and CCB21H (its
hha derivative). The expression of the lacZ gene
was determined by measuring the -galactosidase activities of
cultures growing in lactose-containing LB either without NaCl or with
0.5 M NaCl. Samples were taken when the cultures reached an
OD600 of 0.5. The results obtained and expressed as Miller
units (20) for cultures grown at low and high osmolarities were, respectively, as follows: 324 (strain CCB21) and 381 (strain CCB21H) and 564 (strain CCB21) and 329 (strain CCB21H). Differences between the strains were important only under high-osmolarity conditions. Under such conditions, the -galactosidase activity of
the hha mutant strain was only 58% the activity of the
parental strain. This result suggests lower levels of cAMP in strain
CCB21H caused by the lower expression of enzyme IIAGlc.
To support this notion, we tested if the addition of cAMP to the
culture medium could restore
-galactosidase activity in
the
hha mutant. With this aim, both strains were grown in the
same two culture media already described. At an OD
600 of
0.5,
samples were taken for
-galactosidase measurements (time zero),
and cAMP was added to a final concentration of 3 mM. Thereafter,
the
-galactosidase activities of both strains were monitored
for 1 h. The results obtained are shown in Fig.
5. cAMP addition
allowed the
hha mutant strain growing at high osmolarity to gradually
increase its
-galactosidase activity to a level similar to that
of
the parental strain. At low osmolarity, and as expected from
the
results indicated above, the enzyme activities were already
similar in
both strains before cAMP was added, so no restoration
of
-galactosidase activity was expected.
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FIG. 5.
Effect of the addition of cAMP on the expression of
-galactosidase from strains CCB21 and CCB21H. Cells were grown in LB
medium with 0.5 M NaCl and in LB medium without NaCl. When the cultures
reached an OD600 of 0.5, samples were taken for
-galactosidase measurements (time zero), and cAMP was immediately
added to the cultures. Additional samples were then taken for
-galactosidase measurements at the indicated times. Each bar
represents the relative activity determined as the ratio of the
-galactosidase activities (Miller units) of the hha
(CCB21H) and hha+ (CCB21) strains.
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We must point out that the slight decrease in the relative activities
that can be observed after 20 min was not due to a decrease
in the
enzyme activity of the mutant strain but was due to a slight
increase
in the enzyme activity of the parental strain. This effect
was
presumably caused by the cAMP addition, although we have no
explanation
for it. In any case, the results obtained are consistent
with the
suggestion that the difference detected in the levels
of expression of
lacZ was due to a difference in the levels of
cAMP, which
should occur as a consequence of lower activation
of adenylate cyclase
by enzyme IIA
Glc.
(ii) Effect on inducer exclusion.
It has been reported that
decreasing the amount of enzyme IIAGlc causes cells to
escape from the inducer exclusion process (27). An
experiment designed to monitor effects at the level of inducer exclusion was performed with strains CCB21 and CCB21H growing in
Rich-MOPS containing glucose, a carbohydrate causing this physiological effect. After the wild-type and hha mutant strains were
grown at low and high osmolarities to an OD600 of 0.5, lactose was added to a final concentration of 0.2%. Under such
conditions, determination of the -galactosidase activity after the
addition of lactose is an indirect quantification of the uptake of
lactose and therefore is a measure of the inducer exclusion level. As
no differences between the strains were detected under low-osmolarity
conditions, Fig. 6 shows only the results
obtained at high osmolarity. The addition of lactose caused the
-galactosidase activity of strain CCB21H to increase, while no such
effect was detected in strain CCB21. These results clearly indicate
that the inducer exclusion process was affected by the hha
mutation, as had been anticipated.
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FIG. 6.
Effect of the addition of lactose on the
-galactosidase activities of E. coli CCB21 and CCB21H
grown in Rich-MOPS containing 0.4 M NaCl. When the cultures reached an
OD600 of 0.5 (time zero), lactose was added to a final
concentration of 0.2%. Samples were withdrawn at the indicated times.
Relative -galactosidase activity is the ratio of the
-galactosidase activity (Miller units) measured at the indicated
times to the -galactosidase activity (Miller units) measured just
before the addition of lactose.
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(iii) Effect on the uptake of glucose.
We also examined
whether low levels of enzyme IIAGlc might alter the rate of
uptake of glucose. The rate of transport of glucose in cultures of
strains 5K and Hha-3 growing at low and high osmolarities in Rich-MOPS
containing glucose was therefore determined. The results for cultures
grown at low and high osmolarities were, respectively, as follows: 2.21 (strain 5K) and 1.20 (strain Hha-3) and 1.10 (strain 5K) and 0.35 (strain Hha-3) nmol of glucose min1 mg1.
These data show that alterations in osmolarity affect the rates of
glucose uptake in both strains. Mutant strain Hha-3 showed lower values
than parental strain 5K at both osmolarities, but the largest reduction
in the rate of glucose uptake was detected under high-osmolarity
conditions, in agreement with the suggestion that the lower enzyme
IIAGlc level under such conditions should play a role.
Taken together, all of these results clearly demonstrate that the
catabolite repression regulatory system is affected by the
hha mutation in the same way as that expected for a decrease
in
the expression of enzyme IIA
Glc. Interestingly, Ryu and
Garges (
30) found that in vitro transcription
from the
P1a promoter of the
pst operon, which includes
the
crr gene, is dependent upon a supercoiled DNA template.
As the
hha mutation has been shown to cause a decrease in
the degree of supercoiling
of reporter plasmids (
7), it is
tempting to hypothesize that
it is through changes in DNA supercoiling
that the
hha mutation
causes a decrease in the expression of
enzyme IIA
Glc. Alterations in the expression of protein
IIA
Glc could, in turn, influence the expression of other
genes or operons
which belong to the catabolite repression multigene
system and
which were not considered in this
work.
By using as a model the expression of the toxin alpha-hemolysin, we
have been able to show that the Hha protein is an osmolarity-
and
temperature-dependent modulator of hemolysin synthesis (
22).
In addition, we have shown that DNA supercoiling influences hemolysin
expression and that Hha may influence hemolysin expression by
modifying
the level of DNA supercoiling (
7). Here we extend
our
observations about the modulatory role of Hha and describe
new
phenotypes for
hha mutants. The results provided evidence
that many
hha-mediated changes in protein expression are
dependent
on the osmolarity of the growth medium. It is clear that Hha
is
a global modulator and that osmolarity, probably among other
environmental
factors, dictates the range of proteins whose expression
is affected
by Hha. The relationship among osmolarity, Hha, and the
proteins
OmpA and enzyme IIA
Glc remains to be elucidated.
However, it is apparent that for
E. coli cells, adaptation
to high osmolarity and maintenance of adequate
levels of certain
proteins, such as OmpA and enzyme IIA
Glc, require the
participation of global modulators, such as Hha.
Other
well-characterized factors, such as the sigma factor
s
(
23) or the nucleoid-associated protein H-NS (see reference
1 for a review), have been described as playing an
important
role in the adaptation of bacterial cells to the osmolarity
of
the medium. Hha probably belongs to a cascade of modulators whose
combined effects allow
E. coli cells to fine-tune the
control
of the expression of multigene systems in response to
environmental
stimuli.
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ACKNOWLEDGMENTS |
This work was supported by the CICYT (grant PB94-0899) and in
part by grants from the Swedish Medical Research Council, the Swedish
Natural Science Research Council, and the Göran Gustafsson Foundation for Research in Natural Sciences and Medicine. C. Balsalobre was the recipient of an F. I. Fellowship from the Generalitat de Catalunya.
We thank F. C. Neidhardt and the people in his laboratory for
providing their 2-D PAGE protocols and Ulf Henning and York Stierhof for the generous gift of antibodies against OmpA.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Universidad de
Barcelona, Departamento de Microbiología, Ave. Diagonal 645, Barcelona 08028, Spain. Phone: 934021492. Fax: 934110592. E-mail:
fmunoa{at}porthos.bio.ub.es.
Present address: Department of Microbiology, Umeå University,
S-90187 Umeå, Sweden.
| |
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Journal of Bacteriology, May 1999, p. 3018-3024, Vol. 181, No. 10
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
